Phenolic composition of ten plants species used as ethnoveterinary medicines in Omusati and Kunene regions of Namibia

The therapeutic benefits of phenolic compounds found in plants are well known. The purpose of this study was to determine the phenolic content of ten plant species used as ethnoveterinary treatments in Namibia’s Omusati and Kunene regions. The plants of concern were Aloe esculenta, Fockea angustifolia, Boscia albitrunca, Combretum imberbe, Acacia nilotica, Colophospermum mopane, Acacia erioloba, Ziziphus mucronata, Ximenia americana, and Salvadora persica. An LC–MS approach was used to identify the compounds. To analyse high-resolution UPLC-UV/MS, a Waters Acquity ultra-performance liquid chromatograph (UPLC) with a photodiode array detector was connected to a Waters Synapt G2 quadrupole time-of-flight mass spectrometer (MS). The current study identified a total of 29 phenolic compounds. Flavonoids (epicatechin, (-)-Epigallocatechin, and rutin,) were the most abundant, followed by 2R, 3S-Piscidic acid. Methylisocitric acid was found in all species investigated, with the highest quantities in A. esculenta and X. americana leaf extracts. There were differences in composition and quantity of phenolic compounds in aerial and ground sections between species. The overall findings of the present study would act as a standard for subsequent investigations into the pharmacological potentials of plants species utilized as ethnoveterinary remedies. Priority should be given to isolating, purifying, and defining the active compounds responsible for these plants' activity.

Collection and processing of plant materials. All the extracts used in this study were gathered in Namibia from plants in their natural environments. Aloe esculenta (leaves), Salvadora persica (stem bark), Fockea angustifolia (roots), Boscia albitrunca (leaf and tuber), Combretum imberbe (root and leaf), Acacia nilotica (gum and branch), Colophospermum mopane (leaf and bark), Ziziphus mucronata (leaf and root), and Ximenia americana (leaf) were among the plant parts harvested. These plant parts were harvested during the dry season, between August and November 2020. Mr Teodor Kaambu from the Directorate of Forestry in Windhoek in cooperation with the Namibian Botanical Research Institute (NBRI) identified the plant species employed in the current study. In taxonomic identification, the "Namibian Plants Red Data Book" 17 was used. The Namibia Botanical Research Institute (NBRI) and international regulations prohibiting the collection of whole plants for identification purposes were followed in the collection of these plant parts. ''No complete plants were used or obtained as specimens for the current investigation. The collection of plant parts was done in accordance with Namibian laws, which are outlined in the instructions for collecting plant specimens from the Namibia Botanical Research Institute (NBRI), as well as with international laws that forbid the gathering of complete plants for identification. To avoid the transfer of pests and diseases from one location to another, as required by both Namibian and international legislation governing the collection and/or movement of plant specimens, collected plant parts were inspected for symptoms of diseases and pests'' . The plant parts were air-dried for at least three weeks at room temperature before being pounded into a fine powder. The specimens were subsequently taken to Stellenbosch University in South Africa for phenolic compound analysis.

Phenolic compound analysis.
To analyse high-resolution UPLC-UV/MS, a Waters Acquity ultra-performance liquid chromatograph (UPLC) with a photodiode array (PDA) detector was connected to a Waters Synapt G2 quadrupole time-of-flight (QTOF) mass spectrometer (MS) as described by Hassan et al. 18 . Negative electrospray ionization was employed with a cone voltage of 15 V, desolvation temperature of 275 °C, desolvation gas at 650 L/h, and the remainder of the MS settings optimized for optimal resolution and sensitivity 19 .
Data was collected by scanning in resolution mode and MSE mode from 150 to 1500 m/z 20 . Two channels of MS data were recorded in MSE mode, one with a low collision energy (4 V) and the other with a collision energy ramp (20-60 V) to obtain fragmentation data. For accurate mass determination, leucine enkaphalin was utilized as the lock mass (reference mass), and the device was calibrated with sodium formate 20 . A Waters HSS T3, 2.1 100 mm, 1.7 m column was used to separate the samples as described by New et al. 21 . The mobile phase comprised 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid as solvent B, with a total injection volume of 2 L.
The gradient began with 100% solvent A for 1 min and then shifted to 28% B in a linear fashion over 22 min. It then moved to 40% B for 50 s, then 1.5 min at 100% B, followed by 4 min of re-equilibration to initial conditions. The column temperature was kept at 55 °C and the flow rate was 0.3 mL/min. Compounds were measured according to a calibration curve created by infusing a variety of catechin standards ranging from 0.5 to 100 mg/L catechin 22 . The chemicals used were formic acid (Merck, SA), acetonitrile 200 and methanol 215 (Romil), Catechin (Sigma Aldrich), Millipore water purification. Data analysis. Data was analysed using Principal component analysis (PCA) analysis was conducted using PAST version 4.02, a software for scientific data analysis with functions for data manipulation, plotting, univariate, and multivariate statistics analysis.

Results
The study revealed the presence of 29 phenolic compounds in ten plants studied, with significant differences between species (Tables 1, 2 and 3). In A. nilotica branch extract for example, the primary compound was 2R,3S-Piscidic acid, while in Z. mucronata leaf extract, the main compound was 3-Methyl. Furthermore, corilagin and pedunculagin were only identified in C. imberbe leaves and root extracts, respectively. Except for A. esculenta, epicatechin was found in all plant species. Methylisocitric acid was identified in all species studied, except for F. angustifolia (root) and A. nilotica (gum) extracts. Dihydrox was identified only in B. albitrunca (leaf), C. imberbe  www.nature.com/scientificreports/ (root), and Z. mucronata (root) extracts. Only C. imberbe (leaf), X. American (leaf), and Z. mucronata (root) extracts contained theogallin. Peduncu, on the other hand, was only identified in extracts from two species: C. mopane (bark) and C. imberbe (leaf and root). The content of phenolic compounds in aerial and ground sections differed substantially. The leaf extract of Z. mucronata, for example, contained beta-Glu compound, whereas the root did not Theogallin, 4,6carboxy (4,6-O-[(1R)-1-carboxyethylidene]-alpha-D-galactose), 4-Fumaryl (4-Fumarylacetoacetic acid), and Aralidioside were found in the root extract of the same plant, but not in the leaf extract. Similarly, stizolobat and 3-Methyl (3-Methylglutaconic acid) were exclusively found in the tuber extract of B. albitrunca, but not in its leaf extract. Each phenolic compound's content in aerial and ground sections of the same species varies significantly. The quantity of (-)-epigallo ((-)-Epigallocatechin) and 3-Methyl (3-Methylglutaconic acid) in Z. mucronata leaf extract were higher than those in its root extract. The leaf extract of C. mopane had more 4,6carboxy (4,6-O-[(1R)-1-carboxyethylidene]-alpha-D-galactose) and 4-Fumaryl (4-Fumarylacetoacetic acid) than the bark extract. Tachioside concentrations in the same species' leaf extracts, on the other hand, were lower than those in the bark extracts.

Discussion
The major phytochemical compounds from plants with antioxidant capabilities are known as phenolics and flavonoids [24][25][26][27] . For instance, the high concentration of the phenolic compound catechins in green tea has been linked to its antibacterial properties 28,29 . Similarly, epicatechin, a phenolic compound, was said to be responsible for H. patens' antibacterial action 30 . The effects of gallic acid on wound healing have also been extensively documented by Pal et al. 31 and Comino-Sanz et al. 32 . The discovery of a high quantity of gallic acid in X. americana may help to explain why this plant is reportedly used in EVM to treat wounds in the current study area. Intriguingly, the current study found evidence of gallic acid, catechin, and epicatechin in the examined species. Both plants are known to be used in EVM to treat a variety of veterinary conditions, including mastitis, wound healing, inflammation, and diarrhoea. The high levels of 2R,3S-Piscidic acid detected in A. nilotica branch extract is compatible with Sulaiman and Balachandran's 33 findings, which asserted that A. nilotica is the greatest source of phenolics. Furthermore, Ali et al. 34 , reported that A. nilotica has a wide variety of biologically active, therapeutically useful compounds that have been used in traditional systems of medicine to treat a variety of ailments. When compared to previous investigations, the current study found Z. mucronata leaf extract to contain higher concentration of 3-methyl (3-Methylglutaconic acid) phenolic compound however researchers Gao et al. 35 and Abdoul-Azize 36 found Table 3. Compounds detected in ten plant species used as ethnoveterinary medicines in Omusati and Kunene regions of Namibia.  38 . Furthermore, Generalić et al. 39 also revealed seasonal fluctuations in phenolic compound content, while Maina et al. 40 discovered phenolic compound alterations at different stages of plant development. The pattern of secondary metabolites in each plant is complex 41 ; it varies by tissue and organ; regular differences can be seen between developmental stages, for example, organs important for survival and reproduction have the highest and most potent secondary metabolites, and between individuals and populations. Lattanzio 8 also asserted that there seems to be a correlation between temperature and the concentrations of phenolic compounds in plant tissues, with lower temperatures being linked to higher concentrations of phenolic compounds. Tak and Kumar 42 claimed that phenolics accumulate in the central vacuoles of guard cells and epidermal cells, as well as subepidermal cells of leaves and shoots, which could explain why Z. mucronata and C. mopane leaves for example contained more 3-Methyl (3 Methylglutaconic acid), (-)-epigallo (-)-Epigallocatechin, 4,6carboxy, and 4-Fumaryl Fumaryl (4-Fumarylacetoacetic acid) than root and bark extracts. Additionally, since Phenylalanine Ammonia Lyase (PAL) is a key enzyme in the biogenesis of numerous phenolic compounds, its enhanced activity at lower temperatures may help to explain the elevated quantities of phenolic compounds in the leaves than in roots 43 . Similar results were found by Del Valle et al. 44 who discovered that S. littorea's response to UV stress includes an increase in the concentration of  www.nature.com/scientificreports/ total phenolic compounds. They also discovered that exposure to UV radiation caused a generalized increase in the concentration of anthocyanin and UV-absorbing compounds in S. littorea. It is interesting to note that most of the phenolic compounds found in the plant species studied have clustered on principal component 1 (PC1). However, no comparable findings could be found in the literature. Due to Namibia's frequent droughts, plants have developed several defence mechanisms to deal with or resist this stress 45 . One of these methods is the production of phenolic compounds (polyphenols) 46 . Several native species of severe habitats manufacture phenolic compounds (or polyphenols) as one of their defence mechanisms against the oxidative damage caused by dehydration 47 . The findings of Albergaria et al. 48 support this, demonstrating that medicinal plants cultivated under severe drought had high amounts of phenolic and flavonoid compounds in the leaves, but those grown under higher irrigation had high levels of phenolic compounds in the seeds. These could explain why the phenolic compounds found in the plant species under investigation varied significantly.
Even though the current study found a remarkable composition of phenolic compounds in the plants under investigation, this does not necessarily mean that the screened plant extracts are effective medications or candidates for drug development. However, it does provide basic information about the plant's phenolic compounds, which are thought to play a significant role in the treatment of veterinary diseases as natural antioxidants, antimicrobials, and antiparasitic agents. More testing of these extracts against pathogenic strains of test organisms will be required to assess the pharmacological efficiency of these plants.

Conclusions
The overall findings of the present study would act as a standard for subsequent investigations into the pharmacological potentials of plants utilized as ethnoveterinary remedies. Priority should be given to isolating, purifying, and defining the active compounds responsible for these plants' activity.

Data availability
This article contains all data generated or analysed during this study.